Alternative Splicing of - PowerPoint Presentation

Slide1

Alternative Splicing of Integrins

Alyssa Schwartz

Group Meeting

11-30-15Slide2

Splicing is an Essential Step in the Central Dogma

Pearson, Prentice HallSlide3

Types of Splice Variations

Claude C.

Warzecha

,

Russ P.

Carstens

. Complex

changes in alternative pre-mRNA splicing play a central role in the epithelial-to-mesenchymal transition (EMT

). Seminars in Cancer Biology. Volume 22, Issues 5–6, October 2012, Pages 417–427

A

D. melanogaster

gene

called

Dscam

potentially has

38,016 splice variantsSlide4

Detection of Splice Variants

PCR – mRNA length of variants is often different

RNA-

Seq – local fragments indicate relative abundance of isoforms

R.N

.

Tamura et al. Cell

type-specific integrin variants with alternative- a chain cytoplasmic

domains. Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10183-10187 1991

Uhlen

et al.

Tissue-based map of the human

proteome

.

2015: 

Vol

. 347 no. 6220 

Slide5

Integrin Protein Structure and Extracellular Modifications

Annemieke

A. de

Melker and Arnoud Sonnenberg.

Integrins

: alternative

splicing. as

a mechanism to regulate ligand binding and integrin signaling events. BioEssays 21:499–509, 1999 John Wiley & Sons, Inc. Slide6

Intracellular Modifications

Mara

Fornaro

, Lucia R. Languino. Alternatively spliced variants: A new view of the integrin cytoplasmic domain.

Matrix

Biology. Volume

16, Issue 4, October 1997, Pages

185–193Arjan van der Flier, Arnoud Sonnenberg. Function and interactions of integrins

. Cell and Tissue Research. September 2001, Volume 305, Issue 3, pp 285-298.Slide7

Alternative Splicing and Oncogenic Transformation

A.

Piekielko-Witkowska

et al. Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors.

PLoS

ONE 5(10): e13690. Slide8

In Vitro “Model” system

S.E

.

LaFlamme, et al. Single Subunit Chimeric Integrins as Mimics and Inhibitors of Endogenous Integrin Functions in Receptor Localization, Cell Spreading and Migration, and Matrix Assembly.

The Journal of Cell Biology,

Volume 126, Number 5,

1994

1287-1298Slide9

Mouse Models

Möröy

and Heyd. The impact of alternative splicing in vivo: Mouse models show the way. RNA

 2007. 13: 1155-1171Slide10

α6B can replace

α

6A in

Mouse Cardiac Development C. Gimond

, 

et al.

Cre-

loxP–mediated Inactivation of the α6A Integrin Splice Variant In Vivo: Evidence for a Specific Functional Role of α6A in Lymphocyte Migration but Not in Heart Development. 1998 // JCB vol. 143 no. 1 253-266Slide11

Integrin Alpha 6 Isoform B Dictates Stem-ness

Hira

Lal Goel, Tatiana Gritsko, Bryan Pursell

, Cheng Chang, Leonard D. Shultz, Dale L. Greiner, Jens

Henrik

Norum, Rune Toftgard, Leslie M. Shaw, and Arthur M. Mercurio. Regulated Splicing of the a6 Integrin Cytoplasmic Domain Determines the Fate of Breast Cancer Stem Cells. Cell Reports 7, 747–761, May 8, 2014

SUM-1315

MDA-MB-435Slide12

α

6A Increases Proliferation and WNT signaling

Jean-François

Groulx

,

Véronique

Giroux, Marco

Beauséjour, Salah Boudjadi, Nuria Basora

, Julie C. Carrier and Jean-François Beaulieu. Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells. Carcinogenesis (2014) 35 (6):1217-1227.doi: 10.1093/carcin/bgu006Slide13

Integrin β1A

vs

Integrin

β1DChristian Baudoin,

Marie-José

Goumans

, Christine Mummery, and

Arnoud Sonnenberg. Knockout and knockin of the β1 exon D define distinct roles for integrin splice variants in heart function and embryonic development. Genes & Dev. 1998. 12:1202-1216

Alexey M. Belkin, S. Francesco Retta, Olga Y. Pletjushkina, Fiorella Balzac, Lorenzo Silengo, Reinhard Fassler, Victor E. Koteliansky, Keith Burridge, and Guido Tarone. Muscle

β1

D Integrin Reinforces the Cytoskeleton–Matrix Link: Modulation of Integrin Adhesive Function by Alternative

Splicing.

J Cell Biol. 1997 Dec 15; 139(6): 1583–1595.

β1

A-CHO

 β1

D-CHO

β1

A-GD25

β1

A-GD25

β1

D-GD25

β1

D-GD25

serum-free medium on

Fn

serum-free medium on

Vitronectin

Serum-free

medium on

Fn

for 30 min

Serum-free

medium on

Fn

for

1 hour

mAb

against human β1 integrin for 2 h

}

}

P

lated

on

Fn

and cultured for 1

daySlide14

Integrin Alpha 4 Cytoplasmic Domain Splicing Inhibits Lung Colonization

Hitomi

Kouro

et al. The

Novel

α4

B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion. June 6, 2014 The Journal of Biological Chemistry, 289,16389-16398Slide15

Integrin Alpha 7 Extracellular Variant Alters Phenotype

S

Schöber

et al. The Role of Extracellular and Cytoplasmic Splice Domains of α7-Integrin in Cell Adhesion and Migration on

Laminins

.

Experimental Cell

Research. Volume 255, Issue 2, 15 March 2000, Pages 303–313Chung-Chen Yao, Barry L. Ziober, Rachel M. Squillace and Randall H.

Kramer. α7 Integrin Mediates Cell Adhesion and Migration on Specific Laminin Isoforms. October 11, 1996 The Journal of Biological Chemistry, 271,25598-25603Slide16

Integrin

α

9 Lacking

I

ntracellular

D

omain

Creates Full Length α9 Adhesion Dependence

Shigeyuki Kon, Amha Atakilit, Dean Sheppard. Short form of α9 promotes α9β1 integrin-dependent cell adhesion by modulating the function of the full-length α9 subunit. Experimental Cell Research. Volume 317, Issue 12, 15 July 2011, Pages 1774–1784Slide17

Splicing Structural Changes alter Ionic Activation

Thomas A. Bunch, Timmy L. Kendall, Kishore

Shakalya

,

Daruka

Mahadevan

, Danny L. Brower. Modulation of Ligand binding by alternative splicing of the αPS2 integrin subunit. Journal of Cellular

Biochemistry. Volume 102, Issue 1. 1 September 2007. Pages 211–223Slide18

Conclusions

Alternative Splicing is a complex mechanism that increases the number of human functional isoforms up to 5 fold

Extracellular Integrin splicing modifies binding affinity

Intracellular integrin

splicing alters

downstream signaling